Microbial fermentation culture medium and culture method for increasing yield of gamma-aminobutyric acid

Abstract

The invention belongs to the technical field of culture media, and particularly relates to a microbial fermentation culture medium for improving the yield of gamma-aminobutyric acid, which comprises the following components by concentration: 10-12g / L of glucose, 5-7g / L of sodium succinate, 14-16g / L of yeast extract powder, 14-16g / L of tryptone, 15g / L of sodium glutamate and an ion additive, wherein the ion additive is added according to the condition that every 20mL of the culture medium contains 0.6 to 0.9 mL of Ca < 2 + >, 0.4 to 0.6 mL of K < + >, 0.3 to 0.5 mL of P < 3 + >, 0.5 to 0.8 mL of Mg < 2 + > and 0.4 to 0.5 mL of Mn < 2 + >. According to the culture medium, the yield of gamma-aminobutyric acid (GABA) can be remarkably increased, the problem that the yield of traditional fermentation is low is solved, and the large-scale production requirement is met.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of culture medium, and particularly relates to a microbial fermentation culture medium for improving the yield of gamma-aminobutyric acid. BACKGROUND

[0002] Gamma-aminobutyric acid (GABA) is a naturally occurring four-carbon non-protein amino acid, which is the most important inhibitory neurotransmitter in the central nervous system of mammals, accounts for about 30% of synaptic sites, has multiple physiological functions such as improving sleep, regulating mood, assisting bone development, protecting cardiovascular system, and improving cognitive function, and has a wide application prospect in the fields of functional food, health products, medicine and the like. With the increasing demand of consumers for natural nutritional ingredients, the market demand for gamma-aminobutyric acid continues to rise, and the development of an efficient and stable production method of gamma-aminobutyric acid has become a research hotspot at present.

[0003] At present, the production methods of gamma-aminobutyric acid mainly include chemical synthesis method, biological transformation method and microbial fermentation method. The chemical synthesis method has problems such as harsh reaction conditions, many by-products, insufficient product safety and the like, and is difficult to meet the application requirements in the fields of food and medicine; the biological transformation method depends on recombinant glutamate decarboxylase or whole cell catalysis, and has limitations such as easy influence of enzyme activity and limited substrate conversion rate. In comparison, the microbial fermentation method has advantages such as mild reaction conditions, natural and safe product, low substrate cost and simple operation, and becomes the preferred scheme for large-scale production of gamma-aminobutyric acid at present.

[0004] However, the existing microbial fermentation method for producing gamma-aminobutyric acid still has many technical bottlenecks: The culture medium formula optimization is not systematic, the ratio of carbon source, nitrogen source and auxiliary ingredients lacks scientific design, the nutritional requirements of strain growth and gamma-aminobutyric acid synthesis cannot be accurately matched, and the yield is generally low; The role of ion additives is not fully valued, the existing technology mainly adds a certain type of ion, the synergistic effect of multiple ions is not found, and the addition amount lacks precise control, so it is difficult to activate the activity of key enzymes (glutamate decarboxylase) for synthesizing gamma-aminobutyric acid and maintain the metabolic balance of the strain; Therefore, it is urgent to develop a microbial fermentation culture medium and culture method with scientific formula and high yield and high purity of gamma-aminobutyric acid, to solve the core problems such as low yield in the existing technology, and to meet the needs of industrial large-scale production. SUMMARY

[0005] The purpose of the present application is to solve the above-mentioned technical problems, and to provide a microbial fermentation culture medium for improving the yield of gamma-aminobutyric acid.

[0006] Therefore, the present application provides a microbial fermentation medium for improving the production of gamma-aminobutyric acid, which comprises, in terms of concentration, 10-12 g / L of glucose, 5-7 g / L of sodium succinate, 14-16 g / L of yeast extract powder, 14-16 g / L of tryptone, 15 g / L of sodium glutamate, and ion additives; The ion additives comprise, in terms of concentration, 0.6-0.9 mL of Ca 2+ 0.6-0.9 mL of Ca + 0.4-0.6 mL of K 3+ 0.3-0.5 mL of P 2 +0.5-0.8 mL of Mg 2+ 0.4-0.5 mL of Mn.

[0007] Further, the concentration of glucose is 11 g / L, the concentration of sodium succinate is 6 g / L, the concentration of yeast extract powder is 15 g / L, and the concentration of tryptone is 15 g / L.

[0008] Further, the ion additives comprise, in terms of concentration, 0.75 mL of Ca 2+ 0.50 mL of K + 0.40 mL of P 3+ +0.65 mL of Mg 2 0.45 mL of Mn. 2+

[0009] Further, the microbial fermentation medium comprises the following steps: Preparation of the microbial fermentation medium; Adjustment of the initial pH of the medium to 5.5-6.5; Inoculation of the gamma-aminobutyric acid-producing microbial strain at an inoculation amount of 2-3%; Static culture at 37°C for 72-80 h, collection of the fermentation broth, and extraction of gamma-aminobutyric acid.

[0010] Further, the gamma-aminobutyric acid-producing microbial strain is a lactic acid bacterium or a Bacillus strain capable of expressing glutamate decarboxylase.

[0011] Further, the process for extracting gamma-aminobutyric acid comprises the following separation and purification steps: Addition of a flocculating agent and a coagulating agent to the fermentation broth, stirring and flocculation, and filtration to collect the supernatant; Ultrafiltration of the supernatant using a polyamide hollow fiber membrane with a molecular weight cut-off of 5000 Da at a pressure of 0.1 MPa; Decolorization of the ultrafiltrate by passing through a macroporous adsorption resin, ion exchange chromatography using a cation exchange resin column, and gradient elution using 0.5 M ammonia water after washing with deionized water.​ After the eluent is concentrated, ethanol is added to a concentration of 75% for crystallization, recrystallization after cooling, and the crystals are collected by filtration and dried to obtain high-purity gamma-aminobutyric acid.

[0012] Further, the flocculating agent is chitosan, the addition concentration is 150 mg / L, the coagulant is sodium alginate; the stirring condition is: first stirring at 150 rpm for 5 min, and then stirring at 150 rpm for 25 min.

[0013] Further, the macroporous adsorption resin is DA201-CII, the flow rate is controlled to be 3 BV / h during decolorization, and the loading amount is 5 BV; the cation exchange resin is Amberlite 200C, the adsorption condition is that the flow rate is 1 BV / h, and the loading volume is 2-BV, and the adsorption is stopped when the adsorption reaches the breakthrough point.

[0014] Further, the ultrafiltration treatment further comprises two discontinuous diafiltration operations.

[0015] Further, the initial pH is adjusted to 6.0; the inoculation amount is 2.5%; and the culture time is 77 h.

[0016] The beneficial effects of the present application are: 1. The yield of gamma-aminobutyric acid (GABA) is significantly improved, the problem of low yield in traditional fermentation is solved, and the demand for large-scale production is met.

[0017] 2. The product has high purity, the separation and purification process is efficient, and can meet the purity requirements of high-end application scenarios such as food and medicine.

[0018] 3. The fermentation system is stable, the process parameters are clear, and the repeatability is strong, which avoids large fluctuations in yield during production.

[0019] 4. The process operation is simple, the required equipment is conventional, the raw materials are easy to obtain, and it is suitable for large-scale industrial production to reduce the investment cost.

[0020] 5. The innovation is outstanding, the limitations of traditional single-factor optimization are broken through by multi-component synergistic optimization, and the technical advantages are obvious.

[0021] 6. The production process is safe, the strain and the reagents used do not produce toxic and harmful substances, and the product has wide application prospects. DETAILED DESCRIPTION

[0022] The technical solutions in the embodiments of the present application will be described below. Obviously, the described embodiments are part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art belong to the scope of protection of the present application.

[0023] ​It should be noted that in this application, the terms "comprising", "including", or any other variant thereof are intended to cover non-exclusive inclusion, so that processes, methods, articles or devices including a series of elements not only include those elements, but also include other elements not explicitly listed, or further include elements inherent to such processes, methods, articles or devices. Without more limitations, the element defined by the statement "comprising a" does not exclude the presence of additional identical elements in the process, method, article or device including the element. In addition, it should be pointed out that the scope of the methods and devices in the embodiments of the present application is not limited to performing functions in the order shown or discussed, but can also include performing functions in a substantially simultaneous manner or in reverse order according to the functions involved, for example, the described method can be performed in an order different from that described, and various steps can also be added, omitted or combined. In addition, the features described with reference to certain examples can be combined in other examples.

[0024] A microbial fermentation medium for improving the production of γ-aminobutyric acid, comprising, in terms of concentration: glucose 10-12 g / L, sodium succinate 5-7 g / L, yeast extract 14-16 g / L, tryptone 14-16 g / L, sodium glutamate 15 g / L, and an ionic additive; The ionic additive is added in an amount of Ca2+ 0.6-0.9 mL, K+ 0.4-0.6 mL, P3+ 0.3-0.5 mL, Mg2+ 0.5-0.8 mL, and Mn2+ 0.4-0.5 mL per 20 mL of the medium.

[0025] In the examples of the present application, the concentration of glucose is 11 g / L, the concentration of sodium succinate is 6 g / L, the concentration of yeast extract is 15 g / L, and the concentration of tryptone is 15 g / L.

[0026] In the examples of the present application, the ionic additive is added in an amount of Ca2+ 0.75 mL, K+ 0.50 mL, P3+ 0.40 mL, Mg2+ 0.65 mL, and Mn2+ 0.45 mL per 20 mL of the medium.

[0027] A microbial fermentation method for improving the production of γ-aminobutyric acid, using a microbial fermentation medium, comprising the following steps: Preparation of a microbial fermentation medium; Adjusting the initial pH of the medium to 5.5-6.5; Inoculating a γ-aminobutyric acid-producing microbial strain at an inoculation amount of 2-3%; Static culture at 37°C for 72-80 h, collecting the fermentation broth, and extracting γ-aminobutyric acid.

[0028] In the examples of the present application, the gamma-aminobutyric acid-producing microbial strain is a lactic acid bacteria or a bacillus capable of expressing glutamate decarboxylase.

[0029] In the examples of the present application, the process for extracting gamma-aminobutyric acid comprises the following separation and purification steps: A flocculating agent and a coagulant aid are added to the fermentation broth, and after stirring and flocculation, the supernatant is collected by filtration; The supernatant is subjected to ultrafiltration treatment using a polyamide hollow fiber membrane with a molecular weight cut-off of 5000 Da under a pressure of 0.1 MPa; After decolorizing the ultrafiltrate by macroporous adsorption resin, ion exchange chromatography is performed using a cation exchange resin column, and after washing with deionized water, gradient elution is performed using 0.5 M ammonia solution; After collecting the eluate, crystallization is performed by adding ethanol to a concentration of 75%, and after cooling, recrystallization is performed, the crystals are collected by filtration and dried to obtain high-purity gamma-aminobutyric acid.

[0030] In the examples of the present application, the flocculating agent is chitosan, and the addition concentration is 150 mg / L; the coagulant aid is sodium alginate; and the stirring conditions are: first stirring at 150 rpm for 5 min, and then stirring at 150 rpm for 25 min.

[0031] In the examples of the present application, the macroporous adsorption resin is DA201-CII, and the flow rate is controlled at 3 BV / h and the loading volume is 5 BV during decolorization; the cation exchange resin is Amberlite 200C, and the adsorption conditions are a flow rate of 1 BV / h and a loading volume of 2-BV, and adsorption is stopped when the breakthrough point is reached.

[0032] In the examples of the present application, the ultrafiltration treatment further comprises two discontinuous diafiltration operations.

[0033] In the examples of the present application, the initial pH is adjusted to 6.0; the inoculum size is 2.5%; and the culture time is 77 h.

[0034] The following describes the implementation process of the microbial fermentation medium and culture method for improving the yield of gamma-aminobutyric acid (GABA) in detail in combination with specific experimental data and technical details: I. Experimental materials and instruments: 1. Experimental strain:

[0035] The gamma-aminobutyric acid-producing microbial strain is a lactic acid bacteria (such as Lactobacillus plantarum) or a bacillus (such as Bacillus subtilis) capable of expressing glutamate decarboxylase, and the strain is used for fermentation experiments after being activated and cultured.

[0036] 2. Experimental reagents: Glucose, sodium succinate, yeast extract powder, tryptone, sodium glutamate, calcium ion source, potassium ion source, phosphorus ion Source, magnesium ion source, manganese ion source, chitosan, sodium alginate, macroporous adsorption resin DA201-CII, cation exchange resin Amberlite 200C, ammonia, ethanol, etc. are all analytical reagents.

[0037] 3. Experimental instruments: Constant temperature incubator, centrifuge, ultraviolet-visible spectrophotometer, pH meter, ultrafiltration device (equipped with polyamide hollow fiber membrane with molecular weight cut-off of 5000 Da), ion exchange chromatography column, rotary evaporator, electronic balance, magnetic stirrer, etc.

[0038] II. Optimization and determination of microbial fermentation medium: 1. Single factor experiment to screen the appropriate parameter range: Take tryptone yeast extract glucose (TYG) medium as the basis (initial composition: glucose 10.00 g / L, sodium succinate 10.00 g / L, yeast extract 2.00 g / L, tryptone 5.00 g / L, sodium glutamate 15.00 g / L, pH natural), by controlling a single variable, explore the influence of each factor on GABA production, and determine the appropriate range.

[0039] (1) Screening of the appropriate range of medium composition: Glucose addition: Set 8.00 g / L, 9.00 g / L, 10.00 g / L, 11.00 g / L, 12.00 g / L five gradients, 2.0% inoculum, 37°C culture for 72h. The results showed that the GABA production reached 4.80 g / L when the glucose addition was 10.00 g / L, and the yield in the range of 10~12 g / L was significantly higher than that of other gradients (P<0.05), so the appropriate range was determined to be 10~12 g / L.

[0040] Sodium succinate addition: Set 3.00 g / L, 4.00 g / L, 5.00 g / L, 6.00 g / L, 7.00 g / L five gradients, other conditions are the same as above. The results showed that the GABA production reached 6.91 g / L when the sodium succinate addition was 6.00 g / L, and the yield in the range of 3~6 g / L showed a significant upward trend, and decreased when it exceeded 6 g / L, so the appropriate range was determined to be 5~7 g / L.

[0041] Yeast extract addition: Set 14.00 g / L, 15.00 g / L, 16.00 g / L, 17.00 g / L, 18.00 g / L five gradients, other conditions are the same as above. The results showed that the GABA production reached 5.71 g / L when the yeast extract addition was 15.00 g / L, and the yield in the range of 14~15 g / L increased, and decreased significantly after 15 g / L, so the appropriate range was determined to be 14~16 g / L.

[0042] Tryptone addition amount: set 14.00 g / L, 15.00 g / L, 16.00 g / L, 17.00 g / L, 18.00 g / L five gradients, other conditions are the same as above. The results show that when the tryptone addition amount is 16.00 g / L, the GABA yield reaches 4.64 g / L, the yield increases in the range of 14-16 g / L, and decreases after 16 g / L, so the appropriate range is determined to be 14-16 g / L.

[0043] (2) Suitable range screening of ionic additives: Based on the above optimized medium, set Five addition amount gradients (0 mL / 20 mL, 0.25 mL / 20 mL, 0.50 mL / 20 mL, 0.75 mL / 20 mL, 1.00 mL / 20 mL), single variable determination of GABA yield.

[0044] : 0.75 mL / 20 mL, the yield reaches 11.01 g / L, and the appropriate range is determined to be 0.6-0.9 mL / 20 mL; : 0.50 mL / 20 mL, the yield reaches 10.38 g / L, and the appropriate range is determined to be 0.4-0.6 mL / 20 mL; : 0.50 mL / 20 mL, the yield reaches 9.85 g / L, and the appropriate range is determined to be 0.3-0.5 mL / 20 mL; : 0.75 mL / 20 mL, the yield reaches 11.12 g / L, and the appropriate range is determined to be 0.5-0.8 mL / 20 mL;

[0045] : 0.50 mL / 20 mL, the yield reaches 11.87 g / L, and the appropriate range is determined to be 0.4-0.5 mL / 20 mL.

[0046] (3) Suitable range screening of culture conditions: Initial pH: set 2.0, 4.0, 6.0, 8.0, 10.0 five gradients, other conditions are the same as above. The results show that when the pH is 6.0, the yield reaches 12.97 g / L, the yield increases in the range of 2-6, and decreases in the range of 6-10, so the appropriate range is determined to be 5.5-6.5; Inoculum size: set 1.0%, 3.0%, 5.0%, 7.0%, 9.0% five gradients, other conditions are the same as above. The results show that when the inoculum size is 3.0%, the yield reaches 12.86 g / L, the yield increases in the range of 1%-3%, and decreases in the range of 3%-9%, so the appropriate range is determined to be 2%-3%;

[0047] Culture time: set 24h, 36h, 48h, 60h, 72h, 84h six gradients, other conditions are the same as above. The results show that the yield reaches 13.72g / L when the culture time is 72h, increases in the range of 24~72h, and decreases after 72h, so the appropriate range is determined to be 72~80h.

[0048] 2. Optimal parameter combination: (1) Orthogonal optimization of medium composition and ion addition: Medium composition orthogonal experiment: adopt Orthogonal experiment, with glucose (9, 10, 11g / L), sodium succinate (5, 6, 7g / L), yeast extract powder (14, 15, 16g / L), and tryptone (15, 16, 17g / L) as four factors and three levels. Range analysis shows that the priority of the factors is glucose content > yeast extract powder content > sodium succinate content > tryptone content, and the optimal combination is glucose 11g / L, sodium succinate 6g / L, yeast extract powder 15g / L, and tryptone 15g / L, at which the GABA yield reaches 9.245g / L.

[0049] Ion addition orthogonal experiment: adopt Orthogonal experiment, with (0.70, 0.75, 0.80, 0.85mL / 20mL), (0.40, 0.45, 0.50, 0.55mL / 20mL), (0.40, 0.45, 0.50, 0.55mL / 20mL), (0.65, 0.70, 0.75, 0.80mL / 20mL), (0.40, 0.45, 0.50, 0.55mL / 20mL) as five factors and four levels. Range analysis shows that the priority of the factors is addition amount addition amount addition amount addition amount addition amount, and the optimal combination is 0.75mL / 20mL, 0.50mL / 20mL, 0.40mL / 20mL, 0.65mL / 20mL, 0.45mL / 20mL, at which the GABA yield reaches 15.763g / L.

[0050] (2) Response surface optimization of culture conditions: Based on Box-Behnken design, taking initial pH (A), inoculation amount (B), culture time (C) as independent variables, and GABA yield as response value, a quadratic regression model was fitted. The model was significant (F=4.23, P=0.0352) and had good fitting degree The priority of factors was culture time (C)>initial pH (A)>inoculation amount (B). The optimal combination was initial pH 6.0, inoculation amount 2.5%, and culture time 77 h, and the predicted GABA yield was 14.7058 g / L.

[0051] 3. Determination of optimal medium formula Based on the above optimization results, the optimal microbial fermentation medium formula was determined as follows: glucose 11 g / L, sodium succinate 6 g / L, yeast extract powder 15 g / L, tryptone 15 g / L, and sodium glutamate 15 g / L. Ion additives were added to each 20 mL of medium as follows: 0.75 mL, 0.50 mL, 0.40 mL, 0.65 mL, 0.45 mL.

[0052] III. Implementation of microbial fermentation culture method 1. Preparation of culture medium According to the above optimal formula, each component was accurately weighed and dissolved in deionized water, and stirred uniformly without additional pH adjustment (the initial pH was suitable for subsequent optimization under natural conditions).

[0053] 2. Fermentation culture (1) Adjust the initial pH of the culture medium to 6.0; (2) inoculate the activated GABA-producing microbial strain at an inoculation amount of 2.5%; (3) place the inoculated culture medium in a 37°C constant temperature incubator for static culture for 77 h, and collect the fermentation broth.

[0054] 3. Isolation and purification of γ-aminobutyric acid (1) flocculation pretreatment: 150 mg / L chitosan flocculant and appropriate sodium alginate coagulant are added to the fermentation broth, stirring at 150 rpm for 5 min, and then stirring at 150 rpm for 25 min, after flocculation, the supernatant is collected by filtration, and the flocculation rate reaches 94.74%; (2) ultrafiltration and diafiltration: the supernatant is subjected to ultrafiltration treatment under the pressure of 0.1 MPa by using a polyamide hollow fiber membrane with a molecular weight cut-off of 5000 Da, and then subjected to two times of discontinuous diafiltration, the GABA recovery rate is increased to 98.62%, and the purity reaches 73.82%; (3) decolorization treatment: the liquid subjected to diafiltration treatment is passed through a macroporous adsorption resin DA201-CII, the flow rate is controlled to be 3 BV / h, and the sample amount is controlled to be 5 BV, the decolorization rate reaches 92.1%, and the GABA recovery rate is 93.46%; (4) ion exchange chromatography: the solution after decolorization is added into a glass column filled with cation exchange resin Amberlite 200C, the adsorption flow rate is controlled to be 1 BV / h, the loading volume is controlled to be 2-BV, the adsorption is stopped when the adsorption reaches the breakthrough point , and after washing with deionized water, the elution is carried out by using 0.5M ammonia water solution gradient; (5) crystallization and drying: the eluate is collected, concentrated by rotary evaporation, then 75% ethanol is added for crystallization, after cooling, 90% warm ethanol is used for recrystallization, the crystal is collected by filtration and dried at 80°C until the weight is constant, and the high-purity GABA crystal is obtained.

[0055] IV. Verification experiment and effect analysis 1. Optimal scheme verification According to the optimal medium formula and the culture method, three groups of parallel experiments are carried out, and the average yield of GABA is measured to be 14.8809 g / L, and the standard deviation is 0.12 g / L, and the relative error with the predicted value is only 1.2%, which indicates that the scheme stability is good. After separation and purification, the purity of GABA reaches 98.58%, which is much higher than the expected standard.

[0056] 2. Comparative experiment (1) Comparison with single-factor optimization scheme: when only the medium composition is optimized, the GABA yield is 9.245 g / L, when only the ion addition is optimized, the yield is 15.763 g / L (without optimizing the culture conditions), and when only the culture conditions are optimized, the yield is 12.97 g / L. The optimal scheme of the application has the synergistic effect of various factors, the yield is stable, and the subsequent purification recovery rate is higher; (2) Comparison with the low-cost raw material replacement scheme: the highest GABA yield of the optimization formula using corn saccharification liquid instead of glucose, corn powder or soybean powder instead of tryptone is 5.36 g / L, which is only 36.0% of the optimal scheme of the application, which confirms the absolute advantage of the scheme in yield.

[0057] V. Conclusion The present application determines the optimal microbial fermentation medium formula and culture method by single factor experiment screening suitable parameter range, combining orthogonal experiment and response surface optimization. The scheme is simple and stable in operation, and through the synergistic regulation of medium composition, ion addition and culture conditions, high yield (14.8809 g / L) and high purity (98.58%) of GABA are realized, and the separation and purification process is adapted to the needs of industrial production, which has important practical application value.

[0058] In summary, the microbial fermentation medium and culture method for improving the yield of gamma-aminobutyric acid (GABA) disclosed in the present application realizes the multiple technical advantages of "high yield, high purity, stability and adaptation to industrialization" of GABA production by optimizing the fermentation system and process parameters, and the specific beneficial effects are as follows: The yield is significantly improved, breaking through the upper limit of traditional fermentation: through single factor experiment screening, orthogonal optimization and response surface design, the medium composition (glucose 11 g / L, etc. optimal combination), ion additive synergistic ratio (optimal addition amount of five ions such as 、 , and culture conditions (pH 6.0, inoculum size 2.5%, culture time 77 h) are precisely matched, so that the yield of GABA reaches 14.8809 g / L, which is much higher than that of single factor optimization (maximum 11.87 g / L) and cheap raw material substitution scheme (maximum 5.36 g / L), solving the core problems of low yield and non-systematic optimization in traditional fermentation.

[0059] High product purity and excellent recovery rate: a ladder type purification process of "flocculation -> ultrafiltration -> diafiltration -> decolorization -> ion exchange -> crystallization" is constructed, combined with precise operations such as chitosan flocculation (flocculation rate 94.74%), DA201-CII resin decolorization (decolorization rate 92.1%), Amberlite200C ion exchange chromatography, etc. Finally, GABA with a purity of 98.58% is obtained, and the recovery rate is improved to 98.62% after two discontinuous diafiltrations, which takes into account the product purity and yield, and meets the stringent requirements of high-end application scenarios such as food and medicine for purity.

[0060] The fermentation system is stable and has strong repeatability: the concentration range of each component of the medium, the ion addition gradient and the parameter interval of the culture conditions are clearly defined, and the experimental verification shows that the variation coefficient of the system in different batches of fermentation is less than 1%, avoiding the problem of large fluctuation of yield caused by fuzzy component ratio and fluctuation of environmental parameters in traditional fermentation, providing stable and reliable technical support for industrial large-scale production.

[0061] The process is suitable for industrialization and easy to operate and control: the fermentation process uses static culture without the need for complex aeration equipment; the parameters of each step in the purification process (such as ultrafiltration pressure 0.1 MPa, elution flow rate 3 BV / h, etc.) are clear, the equipment requirements are conventional, and there is no need for special customization; the raw materials selected are industrial-grade materials such as glucose and tryptone, which are easy to obtain, and the cost is controllable and the supply is stable, which reduces the equipment investment and operation difficulty of industrial production.

[0062] The technical solution is scientific and rigorous, and has outstanding innovation: for the first time, the promoting effect of the synergistic action of five ions on GABA synthesis is systematically verified, and the priority of each factor is determined (such as the order of ion addition > ), which breaks through the technical limitations of single ion addition; at the same time, through response surface optimization, the culture conditions are precisely matched, so that each link of the fermentation system is synergistically enhanced, and the technical solution has significant innovation and advancement.

[0063] The product is safe and has a wide application prospect: lactic acid bacteria or bacillus (such as Lactobacillus plantarum and Bacillus subtilis) that can express glutamate decarboxylase are selected, and the safety of the strains has been recognized by the industry; the fermentation process is mild (37℃, pH 5.5~6.5) and no toxic and harmful substances are produced; the purification process uses natural or conventional reagents such as chitosan and ethanol, without secondary pollution, and the product can be widely used in functional foods, health products, medicine and other fields, and has significant market application value.

[0064] The above describes the embodiments of the present application, and in the case of no conflict, the embodiments in the present application and the features in the embodiments can be combined with each other, the present application is not limited to the above specific embodiments, the above specific embodiments are only illustrative, not limiting, and a person of ordinary skill in the art can make many forms under the inspiration of the present application without departing from the scope of the present application and the protection scope of the claims, all of which belong to the protection of the present application.​

Claims

1. A microbial fermentation culture medium for increasing the yield of γ-aminobutyric acid (GABA), characterized in that, The following are included by concentration: glucose 10-12 g / L, sodium succinate 5-7 g / L, yeast extract 14-16 g / L, tryptone 14-16 g / L, monosodium glutamate 15 g / L, and ionic additives. Among them, the ionic additive contains Ca per 20 mL of culture medium. 2+ 0.6~0.9mL, K + 0.4~0.6mL, P 3+ 0.3~0.5mL, Mg 2 +0.5~0.8mL and Mn 2+ Add 0.4~0.5mL of the ingredient.

2. The microbial fermentation culture medium according to claim 1, characterized in that, The concentrations of glucose, sodium succinate, yeast extract, and tryptone are 11 g / L, 6 g / L, 15 g / L, and 15 g / L, respectively.

3. The microbial fermentation culture medium according to claim 1, characterized in that, The amount of the ionic additive added to each 20 mL of the culture medium is: Ca 2+ 0.75mL, K + 0.50mL, P 3+ 0.40 mL, Mg 2 +0.65mL, Mn 2+ 0.45mL.

4. A method for increasing the yield of γ-aminobutyric acid (GABA) through microbial fermentation, using the microbial fermentation culture medium described in any one of claims 1-3, characterized in that, Includes the following steps: Preparation of microbial fermentation culture medium; Adjust the initial pH of the culture medium to 5.5-6.5; Inoculate with γ-aminobutyric acid-producing microbial strains at an inoculation rate of 2-3%; The fermentation broth was statically cultured at a constant temperature of 37℃ for 72-80 hours, and then γ-aminobutyric acid was extracted.

5. The microbial fermentation culture method according to claim 4, characterized in that, The γ-aminobutyric acid-producing microbial strain is a lactic acid bacterium or Bacillus that can express glutamate decarboxylase.

6. The microbial fermentation culture method according to claim 5, characterized in that, The process for extracting γ-aminobutyric acid includes the following separation and purification steps: Add flocculant and coagulant aid to the fermentation broth, stir to flocculate, and then filter to collect the supernatant. The supernatant was subjected to ultrafiltration using a polyamide hollow fiber membrane with a molecular weight cutoff of 5000 Da at a pressure of 0.1 MPa. After decolorizing the ultrafiltrate with macroporous adsorption resin, it was added to a cation exchange resin column for ion exchange chromatography, washed with deionized water, and then eluted with a gradient of 0.5M ammonia solution. After collecting and concentrating the eluent, ethanol was added to a concentration of 75% for crystallization. After cooling, the crystals were recrystallized, filtered, collected, and dried to obtain high-purity γ-aminobutyric acid.

7. The microbial fermentation culture method according to claim 6, characterized in that, The flocculant is chitosan, with an addition concentration of 150 mg / L, and the coagulant aid is sodium alginate; the stirring conditions are: first stir at 150 rpm for 5 min, then stir at 150 rpm for 25 min.

8. The microbial fermentation culture method according to claim 6, characterized in that, The macroporous adsorption resin is DA201-CII, and the flow rate is controlled at 3 BV / h and the sample loading volume is 5 BV during decolorization; the cation exchange resin is Amberlite200C, and the adsorption conditions are a flow rate of 1 BV / h and a loading volume of 2 BV. Adsorption is stopped when the breakthrough point is reached.

9. The microbial fermentation culture method according to claim 6, characterized in that, The ultrafiltration process also includes two discontinuous percolation operations.

10. The microbial fermentation culture method according to claim 4, characterized in that, The initial pH was adjusted to 6.0; the inoculum size was 2.5%; and the culture time was 77 hours.